We report herein a versatile postsynthetic modification of on-column oligodeoxynucleotides (ODNs) using a copper-catalyzed oxidative acetylenic coupling reaction. Hexamers supported on resins via a methylamino-modified linker were prepared, and on-column modifications of ODNs were examined. ArgoPore resin proved to be the best choice for the modification, and introduction of functional molecules, such as anthraquinone, biotin, and fluorescein, resulted in good yields at not only the 5'-terminal but also the internal T-end of the ODNs. This method is applicable to the modification of 12mer ODN consisting of a random sequence. The resulting ODN9 possessing fluorescein at its 5'-terminal acts as a non-RI primer for primer extension assays using the Klenow fragment.